1) Field of the Invention
A method for nucleic acid analysis including injecting a slight amount out of nucleic acid sample, and a microchip for analyzing nucleic acid which enables such method for nucleic acid analysis, wherein, excluding the slight amount of the nucleic acid sample which is used for analysis, most of an amount of the nucleic acid sample that is not used can be obtained as a pure product which is not contaminated with a fluorescent material.
2) Background of the Invention
Nucleic acids such as DNA or RNA extracted from cells are used for diagnosis and research of diseases, but as the amounts thereof are very small, the amplification of samples is essential for analysis. Such amplification of nucleic acid samples is carried out through polymerase chain reaction (PCR), and it requires repeated temperature control between two or three temperatures, depending on the types of enzymes to be used.
Since the success of DNA amplification and the efficiency of amplification in the PCR are determined by complex factors such as temperature control of each step, the length of primers, base sequences, size of template DNA, etc., analysis procedures for identifying amplification results after PCR are essential. In general, gel electrophoresis using slab-gels such as agarose or acrylamide is employed to identify the amplification results after PCR, but it takes many steps and long time from gel manufacturing to electrophoresis and result checking, and the amplification step of samples and the analysis step thereof are separated from each other so that dilution or loss of the samples during the transfer thereof is likely to occur or it may be easily influenced by external environments.
In order to deal with this, methods of identifying PCR products after the PCR procedures on a microchip have been developed (Fu-Chun Huang et al., Electrophoresis, 2006, 27, 3297-3305; Isabel Rodriguez et al., Electrophoresis, 2003, 24, 172-178); however, as these methods allow only one PCR product to be analyzed at one time, they have a low throughput, and as the PCR products are analyzed through fluorescence detection after the reactions with fluorescent materials inside or outside the chip, purely amplified nucleic acids which are not contaminated with fluorescent materials cannot be obtained.
As described in the above, in the slab-gel mannered nucleic acid analysis using agarose or acrylamide by the prior arts, samples cannot be analyzed in a continuous manner from amplification to analysis, and further, as the samples need to be transferred to slab-gels from amplification machines, they are inevitably exposed to external environments so that there is a high contamination possibility of the samples, and the volumes of the amplified nucleic acids have to be sufficient to enable the analysis of the samples. Meanwhile, the methods for nucleic acid analysis using microchips only show the case where respective samples are amplified and then separated, and there is no case of amplified samples being separated prior to the analysis thereof in a continuous-flow manner. Therefore, they fail to accomplish high-throughput analysis, and there is also an issue that purely amplified nucleic acids which are not contaminated with fluorescent materials cannot be obtained.